Molecular self-assembly is the spontaneous organization of molecules into structurally well-defined arrangements due to non-covalent interactions. The resulting supramolecular structure usually provides nanoarchitectures with very defined macroscopic properties1. As a result, molecular assemblies have attracted much attention in relation to the development of novel materials. In the last decade, molecular self-assembly of biopolymer has shown to play a key role in the discovery and design of biomaterials finding applications in the field of medical technology such as, e.g., regenerative medicine and drug delivery systems2,3. Recently, among the different systems investigated, a new class of ionic self-complementary oligopeptide has attracted a great deal of attention due to their ability to spontaneously self-assemble to form stable macroscopic structure in the presence of monovalent cations4.
A number of peptide molecular self-assembly systems have been designed and developed (Table 1). This systematic analysis provided insight into the chemical and structural principles of peptide self-assembly. These peptides are short, simple to design, extremely versatile and easy to synthesize. Three types of self-assembling peptides have been systematically studied thus far. It is believed additional different types will be discovered and developed in the coming years. This class of biological materials has considerable potential for a number of applications, including scaffolding for tissue repair and regenerative medicine, drug delivery of molecular medicine, as well as biological surface engineering. Similar systems have also been described where these peptide systems undergo self-assembly to form gel with regular β-sheet tapes of well-defined structures5. The self-assembly of peptide nanotubes that allow ions to pass through and to insert themselves into lipid bilayer membrane were also described6,7. Furthermore a number of fascinating biomimetic peptide and protein structures have been engineered, such as helical coil-coils, di-, tri- and tetra-helical bundles8-10. However, their applications for materials science and engineering remain under explored. It is likely that these stable coiled coils will be developed as nanomaterials in the future.
Type I Self-Assembling Peptides
Type I peptides, also called “molecular Lego”, form β-sheet structures in aqueous solution because they contain two distinct surfaces, one hydrophilic, the other hydrophobic. See U.S. Pat. No. 5,670,483. Like Lego bricks that have pegs and holes and can only be assembled into particular structures, these peptides can do so at the molecular level. The unique structural feature of these peptides is that they form complementary ionic bonds with regular repeats on the hydrophilic surface. The complementary ionic sides have been classified into several moduli, i.e. modulus I, II, III, IV, etc., and mixed moduli. This classification is based on the hydrophilic surface of the molecules that have alternating + and − charged amino acid residues, either alternating by 1, 2, 3, 4 and so on. For example, molecules of modulus I have −+−+−+−+, modulus II, −−++−−++, modulus, IV −−−−++++. These well defined sequences allow them to undergo ordered self-assembly, resembling some situations found in well studied polymer assemblies.
Upon the addition of monovalent alkaline cations or the introduction of the peptide solutions into physiological media, these oligopeptides spontaneously assemble to form macroscopic structures that can be fabricated into various geometric shapes11. Scanning EM reveals that the matrices are made of interwoven filaments that are about 10–20 nm in diameter and pores about 50–100 nm in diameter12,13.
The molecular structure and proposed complementary ionic pairings of the Type I peptides between positively charged lysines and negatively charged glutamates in an overlap arrangement represent an example of this class of self-assembling β-sheet peptides that spontaneously undergo association under physiological conditions. If the charged residues are substituted, i.e., the positive charged lysines are replaced by positively charged arginines and the negatively charged glutamates are replaced by negatively charged aspartates, there are essentially no drastic effects on the self-assembly process. However, if the positively charged resides, Lys and Arg are replaced by negatively charged residues, Asp and Glu, the peptide can no longer undergo self-assembly to form macroscopic materials although they can still form β-sheet structures in the presence of salt. If the alanines are changed to more hydrophobic residues, such as Leu, Ile, Phe or Tyr, the molecules have a greater tendency to self-assemble and form peptide matrices with enhanced material strength13.
A number of mammalian cells have been tested and all have been found to be able to form stable attachments with the peptide materials11. Several peptide materials have been used to test for their ability to support cell proliferation and differentiation. These results suggested that the peptide materials can not only support various types of cell attachments, but can also allow the attached cells to proliferate and differentiate. For example, rat PC12 cells on peptide matrices were exposed to nerve growth factor (NGF), they underwent differentiation and exhibited extensive neurite outgrowth. In addition, when primary mouse neuron cells were allowed to attach the peptide materials, the neuron cells projected lengthy axons that followed the specific contours of the self-assembled peptide surface.
The fundamental design principles of such self-assembling peptide systems can be readily extended to polymers and polymer composites, where co-polymers can be designed and produced.
Type II Self-Assembling Peptides
Several Type II peptides are developed as “Molecular Switches” in which the peptides can drastically change its molecular structure. One of the peptides with 16 amino acids, DAR16-IV, has a β-sheet structure at ambient temperature with 5 nm in length but can undergo an abrupt structural transition at high temperatures to form a stable α-helical structure with 2.5 nm length14. Similar structural transformations can be induced by changes of pH. This suggests that secondary structures of some sequences, especially segments flanked by clusters of negative charges on the N-terminus and positive charges on the C-terminus, may undergo drastic conformational transformations under the appropriate conditions. These findings can not only provide insights into protein-protein interactions during protein folding and the pathogenesis of some protein conformational diseases, including scrapie, Huntington's, Parkinson's and Alzheimer's disease, but also can be developed as molecular switches for a new generation of nanoactuators.
The peptides of DAR16-IV (DADADADARARARARA) (SEQ ID NO: 30) and EAK12 (AEAEAEAEAKAK) (SEQ ID NO: 22) have a cluster of negatively charged glutamate residues close to N-terminus and a cluster of positively charged Arg residues near C-terminus. It is well known that all a-helices have a helical dipole moment with a partial negative C-terminus toward a partial positive N-terminus15. Because of the unique sequence of DAR16-IV and EAK12, their side chain charges balance the helical dipole moment, therefore favoring helical structure formation. However, they also have alternating hydrophilic and hydrophobic residue as well ionic self-complementarity, which have been previously characterized to form stable β-sheets. Thus the behavior of this Type II of molecules is likely to be more complex and dynamic than other stable β-sheet peptides. Additional molecules with such dipoles have been designed, studied and confirmed the initial findings.
Others have also reported similar findings that proteins and peptides can undergo self-assembly and disassembly or change their conformations depending on the environmental influence, such as its location, pH change and temperature or crystal lattice packing16-18.
Type III Self-Assembling Peptides
Type III peptides, like “Molecular Paint” and “Molecular Velcro”; undergo self-assembly onto surface rather with among themselves. They form monolayers on surfaces for specific cell pattern formation or to interact with other molecules. These oligopeptides have three distinct features. The first feature is the terminal segment of ligands that incorporate a variety of functional groups for recognition by other molecules or cells. The second feature is the central linker where a variable spacer is not only used to allow freedom of interaction at a specified distance away from the surface but also permit the flexibility or rigidity. The third feature is the surface anchor where a chemical group on the peptide can react with the surface to form a covalent bond. This simple system using Type III self-assembly peptides and other substances to engineer surfaces is an emerging technology that will be a useful tool in biomedical engineering and biology. This biological surface engineering technique will provide new methods to study cell-cell communication and cell behavior11.
Other previously pioneered molecular self-assembly systems through the incorporation of organic linkers for surface anchoring have been developed by George Whitesides and his colleagues1.
TABLE 1Type I self-assembling peptides studied.IonicNameSequence (N → C)ModulusStructure  + − + − + − + −RADA16-In-RADARADARADARADA-cIβ(SEQ IDNO: 1)   + − + − + − + −RGDA16-In-RADARGDARADARGDA-cIr.c.(SEQ IDNO: 2)   + − + −RADA8-In-RADARADA-cIr.c.(SEQ IDNO: 3)   + + − − + + − −RAD16-IIn-RARADADARARADADA-cIIβ(SEQ IDNO: 4)   + + − −RAD8-IIn-RARADADA-cIIr.c.(SEQ IDNO: 5)    − + − + − + − +EAKA16-In-AEAKAEAKAEAKAEAK-cIβ(SEQ IDNO: 6)    − + − +EAKA8-In-AEAKAEAK-cIr.c.(SEQ IDNO: 7)   + − + − + − + −RAEA16-In-RAEARAEARAEARAEA-cIβ(SEQ IDNO: 8)   + − + −RAEA8-In-RAEARAEA-cIr.c.(SEQ IDNO: 9)   + − + − + − + −KADA16-In-KADAKADAKADAKADA-cIβ(SEQ IDNO: 10)   + − + −KADA8-In-KADAKADA-cIr.c.(SEQ IDNO: 11)    − − + + − − + +EAH16-IIn-AEAEAHAHAEAEAHAH-cIIβ(SEQ IDNO: 12)    − − + +EAH8-IIn-AEAEAHAH-cIIr.c.(SEQ IDNO: 13)    − − + + − − + +EFK16-IIn-FEFEFKFKFEFEFKFK-cIIβ(SEQ IDNO: 14)    − + − + − +EFK12-In-FEFKFEFKFEFK-cIβ(SEQ IDNO: 15)    − + − +EFK8-IIn-FEFKFEFK-cIβ(SEQ IDNO: 16)    − − + + − − + +ELK16-IIn-LELELKLKLELELKLK-cIIβ(SEQ IDNO: 17)    − − + +ELK8-IIn-LELELKLK-cIIβ(SEQ IDNO: 18)    − − + + − − + +EAK16-IIn-AEAEAKAKAEAEAKAK-cIIβ(SEQ IDNO: 19)    − − − − + +EAK12n-AEAEAEAEAKAK-cIV/IIα/β(SEQ IDNO: 20)    − − + +EAK8-IIn-AEAEAKAK-cIIr.c.(SEQ IDNO: 21)   + + + + − − − −KAE16-IVn-KAKAKAKAEAEAEAEA-cIVβ(SEQ IDNO: 22)    − − − − + + + +EAK16-IVn-AEAEAEAEAKAKAKAK-cIVβ(SEQ IDNO: 23)   + − + − + −KLD12-In-KLDLKLDLKLDL-cIβ(SEQ IDNO: 24)   + − + − + −KLE12-In-KLELKLELKLEL-cIβ(SEQ IDNO: 25)   + + + + − − − −RAD16-IVn-RARARARADADADADA-cIVβ(SEQ IDNO: 26)    − − − − + + + +DAR16-IVn-ADADADADARARARAR-cIVα/β(SEQ IDNO: 27)   − − − − + + + +DAR16-IV*n-DADADADARARARARA-cIVα/β(SEQ IDNO: 28)     − − − − + + + +DAR32-IVn-(ADADADADARARARAR)-cIVα/β(SEQ IDNO: 29)   +−+−+++++−+−++++EHK16n-HEHEHKHKHEHEHKHK-cN/Ar.c.(SEQ IDNO: 30)   +−+−++ ++EHK8-In-HEHEHKHK-cN/Ar.c.(SEQ IDNO: 31)    − − − − − − − − − −VE20*n-VEVEVEVEVEVEVEVEVEVE-cN/Aβ(NaCl)(SEQ IDNO: 32)    + + + + + + + + + +RF20*n-RFRFRFRFRFRFRFRFRFRF-cN/Aβ(NaCl)(SEQ IDNO: 33)β, β-sheet;α, α-helix;r.c., random coil;N/A, not applicable.The numbers follow the name denote the length of the peptides.*Both VE20 and RF20 are in β-sheet form when they are incubated in solution containing NaCl. They do not self-assemble to form macroscopic matrices.
In another attempt to exploit the intrinsic self-assembly of polypeptides as a new avenue to supramolecular materials, Aggeli et al. have designed different short oligopeptides that self-assemble, in non-aqueous solvent, into long, semi-flexible, polymeric β-sheet nanoptape5. These systems were rationally designed to provide strong cross-strands attractive forces between the side chains such as electrostatic interactions, hydrophobic interactions or hydrogen-bondings. In another study, Ghadiri and co-workers have produced self-assembling nanotubes made from cyclic D,L-α-peptides and cyclic β-peptide. They first showed the evidence that D,L-cyclic peptide subunits (cyclo[-(L-Gln-D-Ala-L-Glu-D-Ala)2-]) (SEQ ID NO: 34) adopt flat, ring-shaped conformations and stack through backbone-backbone hydrogen bonding to form extended cylindrical structures20.